-SulfoBiotics- HSip-1SB21 Product Manual

It has been recognized that hydrogen sulfide (H₂S) has an important role as a physiological active substance for vasodilation, cytoprotection, and modulation of insulin secretion. H₂S is considered as a gaseous molecule such as nitric oxide and carbon monoxide. However, around 80% of the total sulfide exists as hydrogen sulfide anion (HS^-) under physiological condition, since the pKa is about 7. In addition, HS^- easily converts to various biochemical molecules such as persulfides and polysulfides, which react with sulfhydryl moieties in a living body. -SulfoBiotics- HSip-1 is a novel fluorescent probe to detect H₂S selectively and it emits strong green fluorescence when it reacts with H₂S.

1 mg

x 1

 

Store in a cool dark place.

• Purified water
• PBS
• Micropipettes

Preparation of 10 mmol/l HSip-1 stock solution

Add 139 μl of purified water to a tube containing 1 mg of HSip-1 and dissolve it by pipetting.

• Store at -20 °C. The reconstituted solution is stable at -20°C for 1 month.

Detection of hydrogen sulfide by HSip-1

1. HSip-1 stock solution (10 mmol/l) was diluted with PBS to prepare 200 μmol/l HSip-1 working solution.
2. Sodium Sulfide (-SulfoBiotics- Sodium Sulfide (Na₂S), 7.8 mg) were dissolved in 1 ml of de-oxygenated H₂O prepared by bubbling of nitrogen gas (100 mmol/l Na₂S solution).
3. Na₂S solution (100 mmol/l, 20 μl) was added to 980 μL of de-oxygenated H₂O to prepare 2 mmol/l Na₂S solution.
4. Na₂S solution (2 mmol/l,100 μl) was added to 900 μl of de-oxygenated H₂O to prepare 200 μmol/l Na₂S solution.
5. Na₂S solution (200 μmol/l) was diluted with de-oxygenated H₂O to prepare various concentrations of Na₂S solution by serial dilution (200, 100, 50, 25, 12.5, 6.3, 3.2, 0 μmol/l).
6. HSip-1 working solution (200 μmol/l, 350 μl) was added to 300 μl of the Na₂S solutions and mixed using a vortex mixer.
7. The solutions were incubated at room temperature for 30 minutes and 200 μl of the solution were transfered to each well (96-well plate).
8. The fluorescence intensities were measured at 516 nm (λ_ex=491 nm) with a microplate reader.

 

Fig. 3  Fluorescence intensity changes at 516 nm with various concentrations of hydrogen sulfide.

• Experimental example on HeLa cells is available. It can be found by searching “SB21“ on our website.

These products were commercialized under the advisory of Dr. Tetsuo Nagano and Dr. Kenjiro Hanaoka (The University of Tokyo).

1. K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura, and T. Nagano, “Development of a Highly Selective Fluorescence Probe for Hydrogen Sulfide”, J. Am. Chem. Soc., 2011, 133, 18003.